(I) One major focus of my section is to understand how muscarinic acetylcholine and other G protein-coupled receptors (GPCRs) function at the molecular level. (II) More recently, we employed gene targeting technology in mice to study the physiological and pathophysiological roles of the individual muscarinic acetylcholine receptor subtypes (M1-M5 mAChRs). (I) STRUCTURE-FUNCTION ANALYSIS OF GPCRs GPCRs form one of the largest protein families found in nature, and estimates are that about 50% of drugs in current clinical use act on specific GPCRs or on GPCR-dependent downstream signaling pathways. To understand how these receptors function at a molecular level, we have used different mAChRs and vasopressin receptors as model systems. To elucidate the structural changes involved in ligand-dependent GPCR activation, we developed a disulfide cross-linking strategy that allows the formation of intramolecular disulfide cross-links between adjacent Cys residues, with the receptor present in its native membrane environment (in situ!). Disulfide cross-linking experiments revealed that agonist activation of the M3 mAChR is associated with striking structural changes on the intracellular surface of the receptor protein (Han et al., JBC 280, 24870, 2005; Ward et al., submitted). We also showed that the agonist-mediated structural changes occurring on the cytoplasmic receptor surface are accompanied by conformational changes in the immediate vicinity of the ligand binding site located close to the extracellular surface of the transmembrane receptor core (Han et al., JBC, in press). To facilitate structure-function studies, we also expressed different muscarinic and vasopressin receptor subtypes as well as different G protein alpha subunits in yeast. Receptor random mutagenesis, followed by yeast genetic screens, led to the identification of mutant receptors endowed with novel functional properties (Li et al., JBC 280, 5664, 2005). This approach, combined with molecular modeling studies, has led to novel insights into how GPCRs function at the molecular level. (II) GENERATION AND ANALYSIS OF MUSCARINIC ACETYLCHOLINE RECEPTOR KNOCKOUT MICE The precise physiological and pathophysiological roles of the individual mAChRs (M1-M5) are not well understood, primarily due to the lack of receptor subtype-selective ligands. To address this issue, we, in collaboration with Chuxia Deng's lab at NIDDK, used gene targeting technology to generate M1-M5 receptor-deficient mice (KO mice). The M1-M5 mAChR KO mice were then subjected to a battery of physiological, pharmacological, behavioral, biochemical, and neurochemical tests. Many of these studies were carried out in collaboration with other laboratories inside and outside of the NIH. This analysis showed that each of the analyzed mAChR KO lines displayed specific functional deficits, indicating that each mAChR subtype mediates distinct physiological functions (for a recent review, see: Wess, Annu. Rev. Pharmacol. Toxicol. 44, 423, 2004). The following key findings were obtained: Roles of M2 mAChRs in cognition and hippocampal synaptic plasticity M2 mAChR KO mice showed significant deficits in behavioral flexibility, working memory, and hippocampal synaptic plasticity (Seeger et al., J. Neurosci. 24, 10117, 2004)). Since impaired muscarinic cholinergic neurotransmission is associated with Alzheimer's disease and normal aging processes, these findings should be of considerable therapeutic relevance. Roles of M5 mAChRs in cocaine self-administration Cocaine self-administration studies showed that the reinforcing effects of low doses of cocaine were significantly reduced in M5 mAChR KO mice (Thomsen et al., J. Neurosci. 25, 8141, 2005). These findings raise the possibility that centrally active M5 receptor antagonists may become therapeutically useful for the treatment of drug addiction. Roles of M1 and M3 mAChRs in glandular function mAChRs expressed by pancreatic acinar cells play an important role in mediating acetylcholine-dependent stimulation of digestive enzyme secretion. Studies with isolated pancreatic acini prepared from M1/M3 receptor double KO mice showed that cholinergic stimulation of pancreatic amylase secretion is mediated by a mixture of M1 and M3 mAChRs and that other mAChR subtypes do not make a significant contribution to this activity (Gautam et al., JPET 313, 995, 2005). These findings clarify the long-standing question regarding the molecular nature of the mAChR subtypes mediating the secretion of digestive enzymes from the exocrine pancreas. Similarly, studies with gastric glands prepared from M1/M3 receptor double KO mice demonstrated that cholinergic stimulation of pepsinogen secretion is also mediated by a mixture of M1 and M3 mAChRs (Xie et al., Am. J. Physiol. 289, G521, 2005). Role of M3 mAChRs in mediating glucose-dependent insulin release Studies with isolated pancreatic islets prepared from WT and M3 mAChR KO mice showed that activation of islet M3 receptors leads to a pronounced potentiation of glucose-dependent insulin release (Duttaroy et al., Diabetes 53, 1714, 2004). This observation suggests that stimulation of beta-cell M3 receptors may represent a useful approach to boost insulin output in type 2 diabetes. Role of M1 mAChRs in T lymphocyte function Indirect evidence suggests that acetylcholine may also play an important immunoregulatory role by contributing to T lymphocyte activation. Analysis of mAChR KO mice showed that CD8+ T cells derived from M1 mAChR KO mice had a defect in the ability to differentiate into cytolytic T lymphocytes (Zimring et al., J. Neuroimmunol. 164, 66, 2005). This observation provides novel information about CD8+ T cell biology and the role of mAChRs in immune regulation. Miscellaneous functions of mAChR subtypes identified by the use of M1-M5 mAChR KO mice In vivo microdialysis studies demonstrated that M4 mAChR KO mice displayed elevated basal dopamine levels and enhanced dopamine responses to psychostimulants in the nucleus accumbens, suggesting that M4 receptors function as inhibitory muscarinic autoreceptors in cholinergic midbrain afferents innervating dopaminergic mesostriatal neurons (Tzavara et al., FASEB J. 18, 1410, 2004). Studies with neuromuscular preparations derived from M2 mAChR KO mice indicated that initiation of acetylcholine release is achieved by depolarization-mediated relief of a tonic block imposed by presynaptic M2 mAChRs (Parnas et al., J. Neurophysiol. 93, 3257, 2005). Cholinergic mechanisms are known to be involved in the regulation of sleep. Daily amounts of paradoxical sleep were found to be significantly decreased in M3 mAChR KO mice, suggesting that M3 receptors play a role in the modulation of paradoxical sleep (Goutagny et al., Neuropsychobiology 52, 140, 2005). Studies with lung slices prepared from mAChR KO mice indicated that M2-receptor mediated activation of the sphingosine kinase (SPHK)/sphingolipid sphingosine 1-phosphate (S1P) signaling pathway contributes to cholinergic constriction of murine peripheral airways (Pfaff et al., Resp. Res. 6, 48, 2005). Norepinephrine release studies carried out with different peripheral organs prepared from mAChR KO mice led to the surprising observation that M3 mAChRs located on cardiac sympathetic nerve endings can act as inhibitory heteroreceptors (Trendelenburg et al., Br. J. Pharmacol. 145, 153, 2005). Like in other regions of the CNS, acetylcholine can facilitate or depress synaptic transmission in occipital slices of mouse visual cortex. Studies with M1-M5 mAChR KO demonstrated that different mAChR subtypes mediate facilitation or depression of cortical synaptic transmission (Kuczewski et al., J. Physiol. 566, 907, 2005).